The present study investigated regional modifications of glycosylation status, sperm association and functional significance of N-and O-linked glycoproteins in epididymal luminal fluid of the rhesus monkey (Macaca mulatta). The predominant glycoproteins of the epididymal luminal fluid that increase in the extent of glycosylation or unmasking of exposed epitopes in a region-specific, maturation-dependent manner, included those of 150, 116, 68, 64, 58 (N-and O-linked) and 170 kDa (O-linked). The higher expression of 40 (N-linked), 38 (N-and O-linked) and 60, 56 and 33 kDa (O-linked) glycoproteins in the proximal caput epididymal fluid was followed by alteration or reorganization of 60, 38 and 33 kDa (O-linked) glycoproteins in the distal segments of the epididymis. The association of epididymal fluid glycoproteins with maturing spermatozoa was identified by generating polyclonal antiserum against monkey caudal sperm membrane in female albino rabbits. The antiserum crossreacted strongly with 58 and 33 kDa epididymal fluid glycoproteins of monkeys and also reacted with 116, 68, 58, 56 and 33 kDa glycoproteins from Triton X-100 extracts of human spermatozoa, indicating the presence of antigenically related components in both species. The functional significance of epididymal fluid glycoproteins in sperm functions was investigated by raising antiserum against a heavily glycosylated 58 kDa glycoprotein (MEF1) of caudal epididymal fluid, which crossreacted with the Triton X-100 extracts of epididymal spermatozoa of monkey and ejaculated human spermatozoa on immunoblots. In an in vitro micro-sperm agglutination assay, anti-MEF1 serum agglutinated both rat caudal epididymal spermatozoa and human spermatozoa. MEF1 seemed to be involved in fertilization as demonstrated by inhibition of fertility (100%) in female albino rabbits and rats immunized with this protein. A sperm-agglutinating 58 kDa glycoprotein of rhesus monkey epididymis with functional significance in fertility was identified, thus indicating that it is a potential candidate for contraceptive vaccine development.

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The present study reports data on post-translational modifications in the glycosylation status during epididymal passage and significance in fertility of a 33 kDa glycoprotein of rhesus monkey (Macaca mulatta), designated as MEF3 (monkey epididymal fluid protein 3). MEF3 exhibited strong affinity for N-linked a-D-mannose groups and O-linked N-Ac-galactosamine linkages in epididymal fluids and exhibited moderate affinity for N-Ac-glucosaminylated (wheat germ agglutinin), fucosylated (Tetragonolotus purpurea), and N-Ac-galactosamine (peanut agglutinin) residues on more mature corpus and caudal spermatozoa in a maturation-dependent manner on Western blots probed with specific biotinylated lectins. Polyclonal antiserum raised against affinity-purified MEF3 from caudal epididymal fluid (CEF) cross-reacted specifically with CEF and caudal sperm membrane of macaque and with Triton X-100 extract of ejaculated human spermatozoa, suggesting the existence of antigenically related components in both species. The tangled agglutination caused by anti-33 kDa serum of human spermatozoa, along with localization of MEF3 on entire sperm surface of epididymal and testicular sperm of monkey and human spermatozoa, suggest the significance of MEF3 in sperm function. The 100% inhibition of fertility of immunized female rabbits with this protein in vivo and inhibition of human sperm penetration in zona-free hamster eggs in vitro suggests the functional significance of MEF3 in fertility. Together, these results clearly indicate that MEF3 has potential significance as a target for antibodies that inhibit sperm function and fertility.

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